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RESEARCH PAPER
Acute vitreoretinal trauma and inflammation aftertraumatic brain injury in miceLucy P. Evans1,2, Elizabeth A. Newell2, MaryAnn Mahajan3, Stephen H. Tsang4,5,6, Polly J. Ferguson2,Jolonda Mahoney2, Christopher D. Hue7, Edward W. Vogel III7, Barclay Morrison III7,Ottavio Arancio8, Russell Nichols8, Alexander G. Bassuk2 & Vinit B. Mahajan3,9
1Medical Scientist Training Program, University of Iowa, Iowa City, Iowa2Department of Pediatrics, University of Iowa, Iowa City, Iowa3Omics Laboratory, Department of Ophthalmology, Stanford University, Palo Alto, California4Bernard and Shirlee Brown Glaucoma Laboratory and Barbara, Donald Jonas Laboratory of Regenerative Medicine, Columbia University, New
York, New York5Edward S. Harkness Eye Institute, Columbia University, New York, New York6Departments of Ophthalmology, Pathology & Cell Biology, Institute of Human Nutrition, Columbia University, New York, New York7Department of Biomedical Engineering, Columbia University, New York, New York8Department of Pathology & Cell Biology, Taub Institute, Columbia University, New York, New York9Palo Alto Veterans Administration, Palo Alto, California
Correspondence
Vinit B. Mahajan, Byers Eye Institute, Omics
Laboratory, Department of Ophthalmology,
Stanford University, Palo Alto, CA 94304.
Tel: 650 723 6995. Fax: 650 498 1528;
E-mail: [email protected]
Funding Information
VBM and AGB are supported by NIH grants
[R01EY026682, R01EY024665,
R01EY025225, R01EY024698 and
R21AG050437]. VBM is supported by
Research to Prevent Blindness (RPB), New
York, NY. SHT is supported by the Barbara &
Donald Jonas Laboratory of Regenerative
Medicine and Bernard & Shirlee Brown
Glaucoma Laboratory are supported by the
National Institute of Health [5P30EY019007,
R01EY018213, R01EY024698,
R21AG050437], National Cancer Institute
Core [5P30CA013696], the Research to
Prevent Blindness (RPB) Physician-Scientist
Award, unrestricted funds from RPB, New
York, NY, USA. PJF is supported by NIH
R01AR059703. CDH, EWV, and BM were
supported by a Multi-University Research
Initiative from the Army Research Office
(W911NF-10-1-0526). EWV was supported
by a National Defense Science & Engineering
Graduate Fellowship from the Department of
Defense (EWV-2012). OA and RN were
supported by the Dept. of the Army –
USAMRAA (W81XWH12-1-0579).
Abstract
Objective: Limited attention has been given to ocular injuries associated with
traumatic brain injury (TBI). The retina is an extension of the central nervous
system and evaluation of ocular damage may offer a less-invasive approach to
gauge TBI severity and response to treatment. We aim to characterize acute
changes in the mouse eye after exposure to two different models of TBI to
assess the utility of eye damage as a surrogate to brain injury. Methods: A
model of blast TBI (bTBI) using a shock tube was compared to a lateral fluid
percussion injury model (LFPI) using fluid pressure applied directly to the
brain. Whole eyes were collected from mice 3 days post LFPI and 24 days post
bTBI and were evaluated histologically using a hematoxylin and eosin stain.
Results: bTBI mice showed evidence of vitreous detachment in the posterior
chamber in addition to vitreous hemorrhage with inflammatory cells. Subretinal
hemorrhage, photoreceptor degeneration, and decreased cellularity in the retinal
ganglion cell layer was also seen in bTBI mice. In contrast, eyes of LFPI mice
showed evidence of anterior uveitis and subcapsular cataracts. Interpretation:
We demonstrated that variations in the type of TBI can result in drastically dif-
ferent phenotypic changes within the eye. As such, molecular and phenotypic
changes in the eye following TBI may provide valuable information regarding
the mechanism, severity, and ongoing pathophysiology of brain injury. Because
vitreous samples are easily obtained, molecular changes within the eye could be
utilized as biomarkers of TBI in human patients.
240 ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Received: 31 August 2017; Revised: 30
November 2017; Accepted: 1 December
2017
Annals of Clinical and Translational
Neurology 2018; 5(3): 240–251
doi: 10.1002/acn3.523
Introduction
Traumatic brain injury (TBI) is damage to the brain
resulting from an external mechanical force including
rapid acceleration/deceleration, pressure waves due to
blast, crush, and impact or penetration by an object.1 TBI
is the leading cause of death and disability in those under
45 years old2 and is a pervasive public health problem in
both civilian life and on the battlefield, affecting persons
of all ages, races, ethnicities, and socioeconomic status.
Even with an annual incidence of 1.7 million in the Uni-
ted States,3 it has been estimated that as many as one
fourth of persons who sustained a TBI did not seek medi-
cal attention.4,5 Survivors of TBI commonly suffer from
disabling changes in personality, sensorimotor function,
and cognition. The long-term sequelae of TBI can result
in lower quality of life, often with the permanent loss of
one or more physical or mental functions, which can pre-
vent return to the workforce after the injury.
Recently, there has been increased recognition of the
impact of TBI on ocular health and vision. Following
even mild TBI, some form of visual disturbance occurs in
up to 90% of patients.6,7 In cases of severe TBI, there are
fewer systematic reviews of impact on vision and ocular
injury, but significant ocular injury is known to occur. In
cases of abusive head trauma in children, for example, an
ophthalmologic exam routinely occurs given its role in
determining etiology. Findings of retinal hemorrhages
during a fundoscopic examination in a child with brain
injury and unknown mechanism are highly suggestive of
abusive head trauma.8 However, in accidental forms of
severe TBI ocular injuries have also been noted including
hyphema (blood within the anterior chamber), traumatic
cataract, corneal injuries, choroidal ruptures, and intra-
retinal hemorrhage.9 Because the eye exam is not rou-
tinely done regardless of TBI severity, the impact of TBI
on vision and eye health is likely underestimated.
A significant increase in TBI in armed services has been
seen largely due to an increase in blast exposures and
increased survival due to the use of body armor. The
increased prevalence of injuries due to improvised explo-
sive devices (IEDs) in present day U.S. military conflicts
necessitates a deeper understanding of the acute and
long-term issues following blast-induced traumatic brain
injury (bTBI). While penetrating eye injuries from
fragmentation can cause lacerations and readily visible
damage, closed-eye or nonpenetrating ocular injuries are
much more difficult to assess. Additionally, closed-eye
injuries are particularly difficult to identify within the
context of trauma or altered mental status.10,11 A previous
study evaluating 46 combat-veterans who had sustained
documented TBI from blast exposures found that 43% of
patients had evidence of closed-eye injuries upon further
evaluation. Many had normal visual acuity, suggesting
that damage occurred at areas away from the fovea and
patients initially presented without symptoms. This study
found that protective ballistic eyewear reduced the num-
ber of open-eye injuries from projectiles, but was not pro-
tective against closed-eye injury in their study sample.11
As current standards for eye protection seem to be insuf-
ficient to prevent ocular injury and many veterans who
experienced blast injuries have returned to civilian life
without ophthalmic examinations, there is a large poten-
tial for undiagnosed closed-globe injuries with unknown
long-term manifestations.11–13
The retina and the optic nerve extend from the
diencephalon during embryological development, are
comprised of neuronal tissue, and are considered a con-
tinuation of the central nervous system (CNS). The axons
of retinal ganglion cells within the retina come together
to form the optic nerve, which respond to injury similarly
to other CNS axons (e.g., retrograde and anterograde
degeneration of axons, scar formation, myelin destruc-
tion). Additionally, the retina and the CNS are both
immunologically privileged sites as they are protected by
the blood–retina barrier (BRB) and blood–brain barrier
(BBB), respectively, and further protected by anti-inflam-
matory and immunoregulatory mediators.14 The parallels
between the retina and the CNS are further supported by
similar eye manifestations in various neurodegenerative
diseases, such as Parkinson’s disease, Alzheimer’s, multi-
ple sclerosis, and stroke.14,15 Thus, the retina reflects the
brain and spinal cord in terms of tissue structure,
response to injury, and interactions with the immune sys-
tem, allowing retinal manifestations to serve as an easily
accessible surrogate in which to study CNS injury.14
TBI can result from a multitude of forces applied in
many distribution patterns in one of the body’s most
complex organs. To address this heterogeneity, several
preclinical models are used to study TBI that differ in
ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 241
L. P. Evans et al. Vitreoretinal Trauma and Inflammation After TBI
mechanism and severity and allow identification of patho-
physiologic changes that may be unique to a single injury
type or seen across various clinical TBI phenotypes.16
While visual disturbances secondary to TBI have been
documented in patients,6,17,18 the phenotypic changes
found in the eye in mouse models of traumatic brain
injury have not been routinely evaluated.19–21 Similar to
the primary brain injury, however, we would expect that
ocular injuries may vary with different mechanisms of
TBI. Characterization of eye manifestations in multiple
preclinical TBI models will thus provide important infor-
mation regarding mechanism of eye injuries in TBI. Fur-
thermore, as the eye is an extension of the brain, an
improved understanding of the molecular mechanisms
underlying eye damage can provide information vital to
the prevention, evaluation, treatment, and long-term
management of patients suffering from TBI. This study
was designed to evaluate acute histological and pheno-
typic changes observed in the eye from two different
mouse models of TBI (Fig. 1). The blast-induced TBI
(bTBI) model applies a shockwave and acceleration to the
entire head including the eye, causing a direct mechanical
deformation to the eye in addition to acceleration/decel-
eration forces causing traumatic axonal injury in the optic
nerve. The lateral fluid percussion (LFPI) model causes
direct damage to the brain, resulting in diffuse and trau-
matic axonal injury to the optic nerve from shearing
forces from a fluid wave. Both models will also incur sec-
ondary injury following TBI due to the endogenous injury
cascades triggered by trauma.
Methods
Blast-induced traumatic brain injury model
Three- to six-month-old wild-type C57BL/6 mice were
assigned to either the blast-induced traumatic brain injury
model (bTBI) (n = 6) or the sham (n = 4) group. The
bTBI model consisted of a 76-mm diameter shock tube
that was previously described in detail,22,23 with a 25-mm
length driver section pressurized with helium gas and a
1240-mm long driven section.24 The mouse was anes-
thetized with isoflurane, the body secured within a rigid
pipe 15 mm away from the shock tube exit to protect the
Figure 1. TBI mechanism schematic. Single shockwave exposure mimics TBI due to blast injury. A compressed driver gas source was connected
to an adjustable driver section of the shock tube, which was aligned vertically over the mouse placed in a mouse holder (A). No obvious tissue
destruction and no accumulation of inflammatory cells was seen at 24 days post blast injury (B). LFPI model mimics TBI due to blunt force trauma.
A Luer-lock hub surrounding a 3-mm craniectomy is connected to IV tubing which extends from a cylinder filled with physiological saline. Injury is
produced by striking the cylinder with a pendulum dropped from a specific height (C). A large lesion in the cortex and disruption of the BBB
following fluid percussion injury is seen with immunohistochemical staining of mouse IgG 24 h post LFPI (arrowheads in D). LFPI, Lateral fluid
percussion injury; TBI, Traumatic brain injury; BBB, blood–brain barrier.
242 ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.
Vitreoretinal Trauma and Inflammation After TBI L. P. Evans et al.
torso and lungs. A metal nose bar and chin support were
used to minimize motion of the head. Pressure transduc-
ers (Endevco 8530B-1000; Meggitt Sensing Systems,
Irvine, CA) were flush-mounted at the shock tube exit, as
well as inside the mouse holder in close proximity to the
animal torso.22,23 Mice were exposed to a single shock-
wave exposure of 269 � 9.8 kPa peak over pressure,
0.73 � 0.021 msec duration, and 67 � 2.3 kPa-msec
impulse directed at the top of the head. Eyes were open
during the blast exposure and were harvested 24 days
post bTBI. Sham-exposed mice were handled similarly in
all respects except that the shock tube was not triggered
and as a result these animals received no shockwave
exposure.
Lateral fluid percussion injury model
The lateral fluid percussion injury (LFPI) model was
modified from a previously described model.25,26 Four-
month-old wild-type C57BL/6 mice were assigned to
either the LFPI (n = 4) or the sham (n = 3) group. On
the day prior to TBI, a craniectomy was completed using
a hand-held 3-mm outer diameter trephine microdrill
(Research Instrumentation Shop, University of Pennsylva-
nia), located lateral to the sagittal suture and centered
between the bregma and lambda. A Luer-loc hub was
secured to the skull around the craniectomy site and was
used to connect mice to the FPI device (Custom Design
and Fabrication, Virginia Commonwealth University,
Richmond, VA). Mice were allowed to recover overnight,
and on the following day were anesthetized with 3%
isoflurane and attached to the FPI device by the Luer-loc
hub. The FPI device was triggered, generating a transient
fluid pulse that impacted the exposed dura with a magni-
tude of 121.6–152.0 kPa. Control mice underwent sham
injury, including craniectomy and identical treatment
until connection to the FPI device, without the applica-
tion of the pressure pulse. Eyes were harvested 3 days
post TBI.
Acute neurological assessment after LFPI
The mice were disconnected from the device and under-
went acute neurologic assessment by measuring the time
to right after they regained consciousness; the righting
reflex is a biological variable previously shown to corre-
late with injury severity.27 To ensure a consistent level of
injury across each mouse, a Tektronix digital oscilloscope
(TDS460A) was used to record the pressure produced by
the fluid percussion apparatus. Injury severity data mea-
sured by the oscilloscope was validated using the righting
reflex to confirm consistent level of injury. This reflex is
commonly used in FPI to reflect the level of injury
delivery across animals. The sham mice righted under
15 sec, while the LFPI-injured mice had righting times of
45 sec, 2.5 min, 4 min, and 6.5 min. The mice were then
reanesthetized with isoflurane in order to remove the
Luer-loc hub and to suture-close the craniectomy and
injury site.
Eye histology
Mice eyes were enucleated by blunt dissection and fixed
as previously described.28,29 Specimens were fixed with
Excalibur’s Alcoholic Z-Fix (Excalibur Pathology), pro-
cessed to paraffin, and 5-lm sections were placed on
slides (Sakura VIP Processor, AO 812 microtome). Slides
were air-dried and then placed in 60°C oven overnight.
Slides were cooled, deparaffinized to water, and stained
with Gill III Hematoxylin (StatLab Medical) and Eosin Y
Alcoholic (BBC Biochemical). Pupil-optic nerve sections
were processed with hematoxylin and eosin, and standard
images were captured under light microscopy for
review.28,29
Retinal ganglion cell quantification
Whole globe sections were used to evaluate the number
of cells within the retinal ganglion cell (RGC) layer. An
observer was masked for the origin of the sections. To
ensure consistency in regional sampling and quality from
one sample to the next, any sections in which the RGC
layer was not continuous with the exiting optic nerve,
where the RGC layer was disrupted, or processing error
led to poor quality images were excluded prior to quan-
tification by a blinded evaluator. A total of 20 eyes were
evaluated within the blast cohort, resulting in n = 5 and
n = 8 that met inclusion criteria for the sham and blast-
injured groups, respectively. Similarly, 14 eyes were evalu-
ated within the LFPI cohort, resulting in n = 5 and n = 6
for the sham and LFPI-injured groups, respectively.
Results are expressed as mean � SEM and a Student’s t-
test was performed. A value of P < 0.05 was considered
significant.
Brain histology
Hematoxylin and eosin stains were used 24 days post
injury on blast brain sections using standard methods.
Immunohistochemical staining of mouseIgG
Following LFPI, sections were stained for IgG for detec-
tion of blood–brain barrier leakage. Mice were deeply
anesthetized with ketamine/xylazine and transcardially
ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 243
L. P. Evans et al. Vitreoretinal Trauma and Inflammation After TBI
perfused with 0.9% saline followed by 4% paraformalde-
hyde in 0.1 mol/L PBS. Brains were extracted, then
immersed and stored in the same fixative. Brains were
cryoprotected in 30% sucrose solution in PBS prior to
cutting 40 lm sections on a freezing microtome. Sections
were then stored in cryopreservative at 20°C until further
processing. At time of staining, sections were washed with
PBS, incubated in peroxidase blocking solution, washed,
and then blocked in 1.5% serum solution. Staining for
IgG was performed on free-floating sections using anti-
IgG antibody (1:500) for 1 h at room temperature. Sec-
tions were then incubated in strep-HRP. Staining was
developed using DAB.
Results
Blast-induced traumatic brain injury model
Our first model mimics blast-induced traumatic brain
injury (bTBI) resulting from an air shock wave, such as
that produced by an IED, being translated into a pressure
wave within the skull-brain complex.19 A compressed gas-
driven shock tube creates an air shock wave, which simu-
lates blast effects (Fig. 1A). Twenty-four days post bTBI,
there was no notable brain tissue destruction and no
obvious inflammatory cells (Fig. 1B).
Histology of whole globe sections of blast-exposed ani-
mals were compared to control animals. Both the sham
and blast-exposed groups had a normal corneal epithe-
lium, stroma, and endothelium. The anterior chambers
were of normal depth without cells, and the angles did
not show recession (Fig. 2A and B, respectively). The iri-
des showed normal pigmentation without rubeosis or
pupillary membranes, and no cataracts were observed.
In the posterior segment, the ciliary bodies of all blast-
exposed and sham eyes contained normal stroma, pig-
mented and nonpigmented layers, and cilia (Fig. 2A and
B). In blast-exposed eyes there were posterior vitreous
detachments (arrowheads in Fig. 2B and D), vitreous
hemorrhage, and some inflammatory cells (Fig. 2C and
D). The abnormal presence of macrophages were found
within the vitreous hemorrhage (arrows in Fig. 2C and
D). In these eyes, there were foci of photoreceptor degen-
eration and pigmentary changes (Fig. 3B, arrows in D).
There was evidence of detached and degenerating pho-
toreceptor outer segments (Fig. 3E). Activated macro-
phages were noted in blast-exposed eyes within the
degenerating photoreceptor layer, as well (arrowheads in
Fig. 3D). Subretinal hemorrhage also occurred (arrow in
Fig. 3F). The retinal pigment epithelium and choroid
showed normal pigmentation, Bruch’s membranes were
intact, the optic nerve was not avulsed, and there were no
neovascular membranes (Fig. 3F). We did observe a
decrease in the retinal ganglion cell (RGC) count in the
blast-exposed mice (Fig. 4A, P = 0.0002). While the sham
mice had an RGC count of 390.8 � 21.9 (Fig. 4B, n = 5),
the bTBI mice had an RGC count of 260.4 � 12.8
(Fig. 4C, n = 8).
Lateral fluid percussion injury model
We utilized a lateral fluid percussion injury (LFPI) model
to examine the effects of another mechanism of TBI on
the eye. The LFPI model, which applied a brief, high-
pressure fluid wave directly to the exposed dura and brain
tissue, mimics the effects of blunt trauma or inertial
mechanisms such as a fall. A pendulum was released, hit-
ting a saline-filled reservoir, which produced a fluid pres-
sure wave that impacted the dural tissue and brain
parenchyma through a craniectomy (Fig. 1C).16,25,26 As a
result of impact by the fluid wave, there was significant
destruction of cortical tissue and disruption of the blood–brain barrier (BBB) at the injury site as detected by
immunohistochemistry staining for mouse IgG 24 h post
LFPI (arrows in Fig. 1D).
Histology of whole globe sections of LFPI-exposed ani-
mals were compared to control animals. Both the sham
and LFPI-exposed groups had a normal corneal epithe-
lium, stroma, and endothelium. The irides showed nor-
mal pigmentation without rubeosis or pupillary
membranes (Fig. 5A and B, respectively). Within the LFPI
mice there was anterior chamber exudate (Fig. 5B–F) andthe presence of inflammatory cells (arrowheads in Fig. 5E
and F) when compared to the sham mice (Fig. 5A and
C). This was seen within both the ipsilateral and con-
tralateral eye relative to the injury site. In the posterior
segment, the ciliary bodies of all sham and LFPI-exposed
eyes contained normal stroma, pigmented and nonpig-
mented layers, and cilia. Additionally, we found a subcap-
sular cataract in one of the LFPI mice (arrows in
Fig. 5E). Unlike the eyes of the bTBI mice, both the LFPI
and sham eyes demonstrated a normal posterior retina
with no cellular infiltrates, no subretinal hemorrhage, and
no photoreceptor degeneration. The retinal pigment
epithelium and choroid showed normal pigmentation,
Bruch’s membrane was intact, the optic nerve was not
avulsed, and there were no neovascular membranes. We
did not observe a difference in the RGC count between
sham and LFPI-exposed mice (Fig. 6A, P = 0.3554), with
RGC counts of 382.0 � 18.31 (Fig. 6B, n = 5) and
409.8 � 21.08 (Fig. 6C, n = 6), respectively.
Discussion
The mammalian eye is similar between mice and humans,
with most differences due to the fact that the mouse eye
244 ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.
Vitreoretinal Trauma and Inflammation After TBI L. P. Evans et al.
is smaller, has a larger lens, and the retina does not have
a fovea. While humans do have higher visual acuity, the
mouse eye is the main animal model for human eye dis-
eases.29 A histological evaluation of mouse eyes post TBI
allowed us to identify several associated ocular injuries
capable of causing long-lasting visual dysfunction. After
sustaining a blast injury, posterior vitreous detachment
and vitreous hemorrhage with macrophages was noted.
bTBI mice also showed foci of photoreceptor degenera-
tion and subretinal hemorrhage. Again, we found abnor-
mal cells along the edge of the inner nuclear layer that
are believed to be activated macrophages. A reduction in
the cellularity of the RGC layer was noted in the bTBI
mice as well. A different phenotype was found following
our second model of TBI, the lateral fluid percussion
injury. These mice showed a large amount of anterior
Figure 2. Posterior vitreous detachment and hemorrhage due to bTBI. Both the sham (A) and blast-exposed (B) mice had anterior chambers of
normal depths without cells, and the angles did not show recession. Vitreous detachment in the posterior chamber was seen in bTBI mice
(arrowheads in B, D) in addition to vitreous hemorrhage with some inflammatory cells (C, D). The abnormal presence of macrophages was noted
as well (arrows in C, D). Hematoxylin and eosin stain. bTBI, blast traumatic brain injury.
ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 245
L. P. Evans et al. Vitreoretinal Trauma and Inflammation After TBI
chamber exudate and debris with some inflammatory
cells, representing an anterior uveitis. Although bTBI mice
showed evidence of retinal ganglion cell loss, the LFPI
mice did not show a difference in retinal ganglion cell
counts. Of note, RGC loss was evaluated just 3 days fol-
lowing LFPI compared to 24 days post blast. In
Figure 3. Subretinal hemorrhage and photoreceptor degeneration due to bTBI. Retinal cross-section of sham (A, C) and blast-exposed (B, D, E, F)
mice. Twenty-four days post bTBI there was evidence of photoreceptor layer degeneration (ONL, OS) as well as loss of the outer plexiform layer
(OPL) (B, arrows in D); some areas saw detachment and partial degeneration of the photoreceptor outer segments (arrows in E). The presence of
activated macrophages were observed around the edge of the inner nuclear layer (INL, arrowheads in D). bTBI also induced subretinal
hemorrhage (arrow in F). Hematoxylin and eosin stain. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer
plexiform layer; ONL, outer nuclear layer; OS, photoreceptor outer segments; bTBI, blast traumatic brain injury.
246 ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.
Vitreoretinal Trauma and Inflammation After TBI L. P. Evans et al.
unpublished data, however, we also saw no difference in
RGC counts 24 days post FPI which is consistent with
what others have reported post FPI.30 The early retinal
cell death in the blast model is striking, since reduction
in retinal ganglion cell layer cellularity and damage to the
optic nerve has been observed at later time points, such
as after 10 months post bTBI.21 A longitudinal evaluation
of our models could help us determine the long-term
effects of LFPI and bTBI on the retinal ganglion cell layer.
Veterans involved in blast-related injuries have seen a
chronic progression of visual symptoms following
closed-globe injuries causing optic nerve and retinal
damage, suggesting an ongoing neurodegenerative
response over time post blast.9,11,31 Within the eye, blast
trauma can cause injuries such as hyphema, retinal
detachment, retinal edema, traumatic optic neuropathy,
and loss of visual field.9,11,13,32 Another mouse model of
closed-globe blast trauma directly applied to the eye saw
a similarly delayed decrease in visual acuity, potentially
due to the gradual increase in oxidative stress, neuroin-
flammation, and focal cell death.33 This delay in the
progression of cell injury and death warrants further
investigation, as early interventions could greatly
improve visual outcomes for patients experiencing bTBI.
While we did not assess visual acuity in the bTBI mice,
the loss of the photoreceptor layer observed would ulti-
mately impair vision and this photoreceptor degenera-
tion could mirror one of the mechanisms leading to
vision loss seen in some human blast injuries.9 Addition-
ally, photoreceptor damage and degeneration after direct
blunt ocular trauma has been well documented in
human34,35 and animal models.36–38
Our method of whole globe histological analysis
revealed an acute anterior uveitis, a finding not previously
reported in studies utilizing the FPI model where instead
delayed axonal swelling and damage to the optic nerve
has been documented, as well as disruption of the blood–brain barrier (BBB).26 We did see BBB damage 24 h post
LFPI, demonstrated by the immunohistochemical staining
of mouse IgG at the injury site; additionally, a loss of
BBB integrity using the bTBI model occurring within the
24 h post injury has been previously well established.23,39
There was no visible disruption of the brain architecture
at 24 days post blast injury. It has been well documented
that the integrity of BBB is reestablished 24 h post blast
injury,23,39but we wanted to verify that overt tissue dis-
ruption did not occur. Damage to the BBB in both of
these models leads to the loss of the brain’s normal
immunologic privilege and isolation from the systemic
immune system. This can lead to secondary injury due to
increased inflammation, and the brain itself can produce
immunologic mediators, contributing to immune dysreg-
ulation and the production of local tissue damage.40
Numerous systemic inflammatory conditions are associ-
ated with anterior uveitis,41 and uveitis frequently coin-
cides with CNS diseases stemming from inflammatory,
infections, and neoplastic processes.42 In the absence of
direct trauma to the eye, uveitis has not been causally
linked to TBI, however, it is possible the aberrant
immune over activation after injury could contribute to
the development of the inflammatory exudate we found
in the anterior chamber of our LFPI mice. While the
LFPI-injured mice demonstrated various righting times,
suggesting different levels of injury severity, we did not
see a correlation between LFPI injury severity and ocular
pathology. However, this study was not powered to deter-
mine if a dose response is present within the ocular injury
we observed.
Figure 4. RGC count decreased in bTBI. A significant decrease was observed in the bTBI mice (A, P = 0.0002). While the uninjured sham mice
had an RGC count of 390.8 � 21.9 (B, n = 5), the bTBI mice had an RGC count of 260.4 � 12.8 (C, n = 8). Eyes were assessed by a masked
observer and were excluded if they did not meet inclusion criteria to ensure consistent regional sampling and quality. RGC, Retinal ganglion cell;
bTBI, blast traumatic brain injury.
ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 247
L. P. Evans et al. Vitreoretinal Trauma and Inflammation After TBI
Figure 5. Anterior chamber exudate due to LFPI. Anterior chamber exudate and the presence of inflammatory cells was noted in the LFPI-
exposed mice (B, D, E) when compared to the sham (A, C). A subcapsular cataract was identified (arrows in E) and abnormal inflammatory cells
were found along the outer surface of the lens and beneath the cornea (c) in LFPI-exposed mouse eyes (arrowheads in E & F). Hematoxylin and
eosin stain. CB, cilliary body. LFPI, lateral fluid percussion injury.
248 ª 2018 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.
Vitreoretinal Trauma and Inflammation After TBI L. P. Evans et al.
As brain trauma may occur through several different
mechanisms, identification of biomarkers that correlate
with injury type, severity, and outcome would facilitate the
ease of diagnosis as well as the translational aspect of devel-
oping and evaluating the efficacy of novel therapies. Using
the eye as a continuation of the CNS, potential biomarkers
identified within the eye could be utilized to reflect what is
happening within the brain, as fluid samples from the vitre-
ous are easily obtained.43–46 Additionally, advances in ocu-
lar imaging techniques could lead to noninvasive diagnosis
of CNS injury. Many neurodegenerative diseases often have
ophthalmic findings and imaging using optical coherence
tomography (OCT) has been proposed as a way to aid in
diagnosis as well as monitoring progression of diseases that
affect both the CNS and the eye.15
Immediately following TBI, eye health is not the pri-
mary concern for physicians and vitreoretinal trauma
may be overlooked. However, the varied phenotypes we
found in this study suggest that TBI may be accompanied
by several types of eye injury. This data in combination
with the high rate of visual symptoms reported after even
mild TBI6,7 suggest that a comprehensive ocular evalua-
tion should be done for all patients following TBI.
Future studies should include histological evaluation of
mice subjected to multiple TBI models across a rigorous
time course, considering the presence of a dose response
to TBI, as well as analyzing vitreous samples to screen for
markers that can be utilized clinically, allowing the eye to
serve as an early indicator of brain trauma. Additionally,
to fully develop this work as a model for TBI-induced eye
injury, we would need to correlate histological changes
with brain function and behavioral studies. Our work
suggests that studies of the eye could have significant
implications on developing and translating new targeted
therapies for brain injury, a clinical problem that cur-
rently lacks effective treatments and results in significant
impairment and decreased quality of life.
Acknowledgments
VBM and AGB are supported by NIH grants
[R01EY026682, R01EY024665, R01EY025225, R01EY024
698 and R21AG050437]. VBM is supported by Research
to Prevent Blindness (RPB), New York, NY. SHT is sup-
ported by the Barbara & Donald Jonas Laboratory of
Regenerative Medicine and Bernard & Shirlee Brown
Glaucoma Laboratory are supported by the National
Institute of Health [5P30EY019007 R01EY018213,
R01EY024698, R21AG050437], National Cancer Insti-
tute Core [5P30CA013696], the Research to Prevent
Blindness (RPB) Physician-Scientist Award, unrestricted
funds from RPB, New York, NY, USA. PJF is supported by
NIH R01AR059703. CDH, EWV, and BM were supported
by a Multi-University Research Initiative from the Army
Research Office (W911NF-10-1-0526). EWV was supported
by a National Defense Science & Engineering Graduate Fel-
lowship from the Department of Defense (EWV-2012). OA
and RN were supported by the Dept. of the Army –USAMRAA (W81XWH12-1-0579).
Conflict of Interest
The authors have no commercial or financial interests
associated with this article.
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